A Deubiquitinating Activity Is Conserved in the Large Tegument Protein of the Herpesviridae

Abstract

The largest tegument protein of herpes simplex virus 1 (HSV-1), UL36, contains a novel deubiquitinating activity embedded in it. All members of the Herpesviridae contain a homologue of HSV-1 UL36, the N-terminal segments of which show perfect conservation of those residues implicated in catalysis. For murine cytomegalovirus and Epstein-Barr virus, chosen as representatives of the beta-and gammaherpesvirus subfamilies, respectively, we here show that the homologous modules indeed display deubiquitinating activity in vitro. The conservation of this activity throughout all subfamilies is indicative of an important, if not essential, function.Modification of proteins by ubiquitin (Ub) plays a pivotal role in a multitude of cellular processes, including proteolysis, cell cycle control, receptor internalization, and sorting within the endo/lysosomal system (7,14,16). Ubiquitination is achieved by an enzymatic cascade comprising a Ub-activating enzyme (E1), several Ub-carrier proteins (E2s), and hundreds of Ub ligases (E3s). Ubiquitination can be reversed by several families of enzymes collectively designated deubiquitinating enzymes (DUBs) (1,15).A number of viruses have evolved strategies to manipulate the ubiquitination status of host cell proteins, both through conjugation and deconjugation (2,4,6,10,13). Recently, we reported the identification of a novel viral ubiquitin-specific protease (USP), UL36 USP , encoded by the herpes simplex virus 1 (HSV-1) genome (9). UL36USP is a polypeptide of approximately 420 amino acids (aa) carried within the N-terminal portion of UL36, the largest tegument protein (3,164 aa) of HSV-1. This activity was detected through the use of mechanism-based, activesite-directed probes and confirmed by expression in Escherichia coli of a corresponding fragment that cleaves ubiquitin-based substrates. UL36USP activity peaks at late stages of viral replication and appears to require proteolytic processing from fulllength UL36 (9). The N-terminal UL36 fragment is well conserved in alphaherpesviruses, and a low homology to corresponding genes of the betaherpesvirus and gammaherpesvirus subfamilies was apparent in sequence alignments, but with strict conservation of the proposed catalytic residues. DUB activity may therefore be well conserved across the herpesvirus family and, if this is proven to be correct, would suggest an important function for this type of activity.We therefore set out to investigate the possible DUB activity of two phylogenetically distant homologues of HSV-1 UL36 USP , each representing a different subfamily of the Herpesviridae. We chose UL36 homologues encoded by mouse cytomegalovirus (MCMV, M48) and Epstein-Barr virus (EBV, BPLF1) as representatives of the beta-and gammaherpesvirus subfamilies, respectively. In order to assess the degree of homology between UL36 from HSV-1 and its MCMV and EBV counterparts, a sequence alignment was generated, covering the first 336 aa (the numbering refers to HSV-1) of UL36 (Fig. 1). Overall, the homology to HSV-1 is rather low, with on...

A herpesvirus ubiquitin-specific protease is critical for efficient T cell lymphoma formation

“…Nonetheless, sequence comparisons across the Herpesviridae show the presence of a few absolutely conserved residues (Cys, Asp, His, Glu), all of which are consistent with involvement in a potential thiol protease active site. We have, meanwhile, confirmed both the mechanism of action of such ubiquitin-based probes (11)(12)(13)(14)(15) and the identity of the viral cysteine protease domain as an authentic USP by crystallographic analysis of the homologous segment of the murine cytomegalovirus (MCMV) M48 protein (16). Notwithstanding the conservation of the identified USP activity in all herpesviruses, we do not know whether this activity makes a contribution to the replicative success and pathogenicity of herpesviruses in vivo.…”

“…So far, only HSV-1 has yielded an enzymatically active, proteolytic fragment that is distinct from the intact tegument protein in virus-infected cells. We cloned and expressed the corresponding region of the MDV U L 36 gene (referred to as MD-V USP ), based on homology and secondary structure predictions (13,14). This 322-residue fragment, when expressed by in vitro transcription/translation, readily formed adducts with Ub-VME in a manner sensitive to the inclusion of N-ethylmaleimide (data not shown).…”

Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

“…For example, the NS1B protein of influenza B virus prevents ISGylation of proteins by binding to ISG15 (37). Like SARS-CoV PLpro, a number of viral proteases have also been shown to deconjugate Ubl modifiers, including proteases from herpes simplex virus 1 and homologues (38,39), African swine fever virus (40), and adenovirus (41). By analogy, these parallel studies strongly suggest the possibility that SARS-CoV uses similar mechanisms of protection against ubiquitination or ISGylation.…”

Dynamic ubiquitination drives herpesvirus neuroinvasion

“…A critical effector of this process is a conserved herpesvirus deubiquitinase (DUB) (a cysteine protease that removes ubiquitin from target posttranslationally modified proteins) (6)(7)(8). The DUB is housed in the amino terminus of the pUL36 protein (protein encoded by the 36th open reading frame in the unique-long segment of the viral genome), amounting to less than 10% of the total mass of this large (>250 kDa) structural component of the virion (9). pUL36 is attached directly to the icosahedral capsid shell of these enveloped viruses (10)(11)(12) and, due to its location between the capsid and envelope, is referred to as a herpesvirus tegument protein.…”